Field of the Invention
The present invention relates generally to novel aromatic nitro and nitroso compounds and their metabolites useful as potent, selective, stable and safe anti-tumor and anti-viral agents, and to a method of treatment of tumorigenic and viral diseases using aromatic nitro and nitroso compounds and their metabolites. More specifically, it relates to the use of various nitro compounds including halo-nitro-estradiol, halo-nitroso-estradiol, halo-nitro-estrone, halo-nitroso-estrone, halo-nitro-estriol, halo-nitroso-estriol, halo-nitro-equilenin, halo-nitroso-equilenin, halo-nitro-equilin, halo-nitroso-equilin, halo-nitro-stilbestrol, halo-nitroso-stilbestrol, 4-halo-3-nitrobenzamide, 4-iodo-3-nitrobenzamide, 4-halo-3-nitrobenzopyrone, 4-iodo-3-nitrobenzopyrone, 5-halo-6-nitro-1,2-benzopyrones, 5-iodo-6-nitro-1,2-benzopyrones, 6-nitro-1,2-benzopyrone, 3-nitrobenzamide, 5-nitro-1(2H)-isoquinoline, 7-nitro-1(2H)-isoquinoline, 8-nitro-1(2H)-isoquinoline, 2-nitrobenzamide, and 4-nitrobenzamide their homologues and salts, in suppressing and inhibiting tumor growth and the growth of certain viruses in a mammalian host.
The State of Art and Related Disclosures
Cancer and viral infections are a serious threat to modern society. Malignant cancerous growths, due to their unique characteristics, pose serious challenges for modern medicine. These characteristics include: uncontrollable cell proliferation resulting in unregulated growth of malignant tissue, an ability to invade local and even remote tissues, lack of differentiation, lack of detectable symptoms and most significantly, the lack of effective therapy and prevention.
Cancer can develop in any tissue of any organ at any age. The etiology of cancer is not clearly defined but mechanisms such as genetic susceptibility, chromosome breakage disorders, viruses, environmental factors and immunologic disorders have all been linked to a malignant cell growth and transformation.
A particularly difficult type of cancer to treat is breast cancer. Approximately 130,000 new cases of breast cancer occur per year in the United States and 70% of these patients will ultimately have recurrence and die of the disease. (Cancer of the Breast, Donegan, W. L., et al. eds., p.504 (1988)).
Antineoplastic chemotherapy currently encompasses several groups of drugs including alkylating agents, purine antagonists and antitumor antibiotices. Alkylating agents alkylate cell proteins and nucleic acids preventing cell replication, disrupting cellular metabolism and eventually leading to cell death. Typical alkylating agents are nitrogen mustard, cyclophosphamide and chlorambucil. Toxicities associated with alkylating agents treatment include nausea, vomiting, alopecia, hemorrhagic cystitis, pulmonary fibrosis and an increased risk of development of acute leukemia.
Purine, pyrimidine and folate antagonists are cell cycle and phase specific and, in order to promote an anti-tumor effect, they require cells to be in the cell replication cycle and in the DNA synthesis phase of replication. The purine antagonists such as 6-mercaptopurine or 6-thioguanidine inhibit de novo purine synthesis and interconversion of purines. The pyrimidine antagonists, such as cytarabine, 5-fluorouracil or floxuridine inhibit DNA synthesis by inhibiting deoxycytidylate kinase and DNA polymerase.
Folate antagonists, eg. methotrexates, bind tightly with the intracellular enzyme dihydrofolate reductase ultimately leading to cell death resulting from an inability to synthesize pyrimidines. Toxicities associated with the use of these compounds include alopecia, myelosuppression, vomiting, nausea, and cerebellar ataxia, among others.
Plant alkaloids such as vincristine, vinblastine or podophyllotoxins etoposide and teniposide generally inhibit mitosis and DNA synthesis and RNA dependent protein synthesis. Toxicities of these drugs are similar to those described above and include myopathy, myelosuppression, peripheral neuropathy, vomiting, nausea and alopecia.
Antitumor antibiotics such as doxorubicin, daunorubicin and dactinomycin act as intercalators of DNA, preventing cell replication, inhibiting synthesis of DNA-dependent RNA and inhibiting DNA polymerase. Bleomycin causes scission of DNA and mitomycin acts as inhibitor of DNA synthesis by bifunctional alkylation. Toxicities of these antibiotics are numerous and severe and include necrosis, myelosuppression, anaphylactic reactions, anorexia, dose-dependent cardiotoxicity and pulmonary fibrosis.
Other compounds used for chemotherapeutical treatment of cancer are inorganic ions such as cisplatin, biologic response modifiers such as interferon, enzymes and hormones. All these compounds, similarly to those mentioned above, are accompanied by toxic adverse reactions.
Endocrine therapy in the treatment of breast cancer is an alternative to chemotherapy. Estrogen is a highly potent mammary mitogen and is the major stimulus for the growth of hormone-dependent breast cancer. Estradiol (an estrogen) is believed to stimulate breast growth (mitogenesis) by binding to nuclear estrogen receptors which stimulate increases in RNA polymerase activity and ultimately cancer cell growth. (Cancer of the Breast, Donegan, W. L., et al. eds., p.504 (1988)).
Thus, it would be extremely advantageous to provide safe and non-toxic chemotherapeutic compounds which would effectively inhibit cancer cell proliferation and suppress neoplastic growth. (The Merck Manual, 1218-1225 (1987), 15th Ed.). In particular, it would be advantageous to provide estrogen-like non-toxic chemotherapeutic molecules that would specifically target breast cancers. Novel compounds of this invention provide such treatment.
Similar to cancer, the high degree of infectiousness and fast reproduction cycle of viruses within host organisms make viruses a nuisance and a health hazard.
There is no simple treatment of viral diseases. Viruses are not susceptible to antibiotics. The only available treatment of viral diseases is chemotherapy utilizing viral replication inhibitors in host cells (The Merck Manual, 170 (1982), 14th Ed.). Examples of these chemical agents are idoxuridine, acyclovir, ribavirin, vidarabine, gancyclovir, adenine arabinoside (ABA-A) and AZT. These, and other viral replication inhibitors, however, are cytotoxic, hepatotoxic, neurotoxic, nephrotoxic and teratogenic (Virus Diseases, 1-6 (1978), Crown Publishers, New York).
Human immunodeficiency virus (HIV) infections known as acquired immunodeficiency syndrome (AIDS), presently constitute a worldwide health hazard. HIV infections are almost always fatal due to a weakened immunoresistance, leading to opportunistic infections, malignancies and neurologic lesions.
There is no effective treatment for AIDS other than the treatment of the opportunistic infections, neoplasms and other complications. Available cytostatic (AZT) and antiviral (acyclovir) drugs are extremely toxic and cause severe adverse reactions.
Thus it would be highly desirable to have available an effective and yet nontoxic treatment of viral diseases, in particular, AIDS.
Herpes simplex virus type-1 and 2 are also wide spread infections. They may occur in AIDS patients as one of the opportunistic infections. Type-1 HSV strain (HSV-1) commonly causes herpes labialism located on a lip, and keratitis, an inflammation of the cornea. Type-2 HSV is usually located on or around the genital area and is generally transmitted primarily by direct contact with herpetic sore or lesions. HSV-2 has been related to the development of uterine cancer.
Herpes simplex virus is very infectious and is rapidly and easily transferable by contact. There is no specific therapy to this extremely painful viral infection. Current treatment of HSV infections is limited primarily to systemic administration of the above-mentioned antiviral drugs with corresponding adverse side affects.
The antiviral agents used for HSV treatment are non-selective inhibitors of HSV replication affecting the replication of normal cells as well. Therefore, when used in doses large enough to inactivate all of the active herpes viruses dormant in the sensory ganglia, these compounds may also be highly disruptive to host cell DNA replication.
Thus, it would be advantageous to have available non-toxic treatment of HSV infections.
Cytomegalovirus (CMV), a dangerous co-infection of HIV, is a subgroup of highly infectious viruses having the propensity for remaining latent in man. CMVs are very common among the adult population and as many as 90% of adults have been exposed to and experienced CMV infections. CMVs are normally present in body liquids such as blood, lymph, saliva, urine, feces, milk, etc. CMV infections may cause abortion, stillbirth, postnatal death from hemorrhage, anemia, severe hepatic or CNS damage. Particularly dangerous are CMV infections afflicting AIDS patients, where CMV may cause pulmonary, gastrointestinal or renal complications. There is no specific therapy for CMVs. Unlike HSV, CMV is resistant to acyclovir, and to other known antiviral drugs.
Thus, it would be extremely advantageous to have available a drug which would effectively inhibit CMV infections.
Recently, a series of highly effective anti-tumor and anti-viral drugs were identified. These drugs include: substituted and unsubstituted 6-amino-1,2-benzopyrones which are the subject of copending U.S. patent application Ser. No. 07/845,342 filed on Mar. 4, 1992, entitled "6-Amino-1,2-Benzopyrones Useful for Treatment of Viral Diseases;" 5-iodo-6-amino-1,2-benzopyrones and 5-iodo-6-nitroso-1,2-benzopyrones which are the subject of U.S. Pat. No. 5,484,951 issued on Jan. 16, 1996 entitled "Novel 5-Iodo-6-Amino-1,2-Benzopyrones and Their Metabolites Useful as Cytostatic and Antiviral Agents" and Ser. No. 08/021,989 filed on Feb. 24, 1993 entitled "Novel 5-Iodo-6-Amino-1,2-Benzopyrones and Their Metabolites Useful as Cytostatic Agents;" 3-nitrosobenzamides, 6-nitroso-1,2-benzopyrones and nitroso-1-(2H)-isoquinolinones which are the subject of copending U.S. patent application Ser. Nos. 07/780,809, 07/893,429 and 07/965,541 filed Oct. 22, 1991, Jun. 4, 1992 and Nov. 2, 1992, respectively, and entitled "Adenosine Diphosphoribose Polymerase Binding Nitroso Aromatic Compounds Useful As Retroviral Inactivating Agents, Anti-retroviral Agents and Anti-tumor Agents;" various halo-nitro compounds which are the subject of copending U.S. patent application Ser. No. 08/060,409 filed May 12, 1993 entitled "Novel Aromatic Compounds and Their Metabolites Useful as Anti-viral and Anti-tumor Agents" the disclosures of which are incorporated herein by reference.
These drugs are of remarkably low toxicity, yet highly effective inhibitors of tumor and viral replication in cell cultures and in human blood. Their therapeutic spectrum appear to be particularly useful for suppression and inhibition of cancer growth and for treatment of the most dangerous viral infections, such as AIDS and herpetic infections.
The mechanisms of action of the the C-nitroso drugs have been recently elucidated. Recently published experiments have shown that aromatic C-nitroso ligands of poly (ADP-ribose) polymerase preferentially destabilize one of the two zinc fingers of the enzyme coincidental with a loss of enzymatic activity but not DNA binding capacity of the protein (Buki, et al., FEBS Lett. 290:181-185 (1991)). Based on the similarity to results obtained by site-directed mutagenesis (Gradwohl, et al., Proc. Natl. Sci. USA 87:2990-2992 (1990)), it appears that the primary attack of C-nitroso ligands occurred at zinc finger FI (Buki, et al., FEBS Lett. 290:181-185 (1991)). 6-nitroso-1,2 benzopyrone (6-NOBP) and 3-nitrosobenzamide (3-NOBA), two C-nitroso compounds that inactivate ADPRT at one zinc finger site completely suppressed the proliferation of leukemic and other malignant human cells and subsequently produced cell death. Tumoricidal concentrations of the drugs were relatively harmless to normal bone marrow progenitor cells and to superoxide formation by neutrophil granulocytes. The cellular mechanisms elicited by the C-nitroso compounds consists of apoptosis due to DNA degradation by the nuclear calcium/magnesium dependent endonuclease (Rice et al. Proc. Natl. Sci. USA (1992) 89:7703-7707). This endonuclease is maintained in a latent form by poly(ADP-ribosyl)ation, but inactivation of ADPRT by C-nitroso drugs derepresses the DNA-degrading activity. Therefore, the C-nitroso compounds are effective anti-tumor compounds.
Retroviral nucleocapsid (NC) proteins and their respective gag precursors from all strains of known retroviruses contain at least one copy of a zinc-binding polypeptide sequence of the type Cys-X2-Cys-X.sub.4 -His-X.sub.4 -Cys (CCHC) (Henderson, et al., Biol. Chem. 256:8400-8406 (1981)), i.e., a zinc finger domain. This CCHC sequence is essential for maintaining viral infectivity, (Gorelick, et al., Proc. Natl. Acad. Sci. USA 85:8420-8424 (1988), Gorelick, et al., J. Virol. 64:3207-3211 (1990)), therefore, it represents an attractive target for viral chemotherapy. The HIV-1 gag proteins function by specifically binding to the HIV-1 RNA, anchoring it to the cell membrane for budding or viral particles (Meric, et al., J. Virol. 63: 1558-1658 (1989) Gorelick, et al., Proc. Natl. Acad. Sci. USA 85:8420-8424 (1988), Aldovini, et al., J. Virol. 64:1920-1926 (1990), Lever, et al., J. Virol. 63:4085-4087 (1989)). Site-directed mutagenesis studies demonstrated that modification of Cys or His residues results in defective viral RNA packaging and noninfectious viral particles are formed (Aldovini, et al., J. Virol. 64:1920-1926 (1990), Lever, et al., J. Virol. 63:4085-4087 (1989)).
Based on the occurrence of (3Cys, 1His) zinc binding sites in both retroviral nucleocapsid and gag-precursor proteins and in poly(ADP-ribose) polymerase it was reasoned that C-nitroso compounds may also have anti-retroviral effects. Recently it was demonstrated that 3-NOBA and 6-NOBP inhibit infection of human immunodeficiency virus HIV-1 in human lymphocytes and also eject zinc from isolated HIV-1 NC zinc fingers and from intact HIV-1 virions. The zinc-ejected HIV-1 virions exhibit complete loss of infectivity in human lymphocytes. Therefore, the C-nitroso compounds are effective anti-viral compounds.
While these C-nitroso compounds have been found to be quite effective in preliminary in vitro tests, they are relatively water insoluble at physiological pH, exhibit limited stability and limited predictability of delivery to the affected cells due to their solubility and stability characteristics. It is thus of interest to identify stable precursor molecules to serve as pro-drugs for the active C-nitroso compounds, especially the aromatic C-nitroso compounds. These pro-drugs would be converted to the active C-nitroso compounds in vivo. Ideally, these pro-drugs would be stable and soluble for convenient use as anti-retroviral and anti-cancer compositions.
Halo nitro compounds are reduced in vivo to active halo nitroso compounds and therefore provide a ready source of in vivo anti-tumor and anti-retroviral C-nitroso compounds.
4-iodo-3-nitrobenzamide (4-I-3-NO.sub.2 BA) has not been hitherto known or described. Three structural isomers of 4-I-3-NO.sub.2 BA are known in the literature: 2-iodo-4-nitrobenzamide, 2-iodo-5-nitrobenzamide and 3-iodo-5-nitrobenzamide (Chem Abstracts: 101: 230049f (1983), 24:3367 (1930) and 97:23081n (1982). No medical use was reported for the compounds.
Halo nitro and nitroso estrogen compounds including halo-nitro-estradiol, halo-nitroso-estradiol, halo-nitro-estrone, halo-nitroso-estrone, halo-nitro-estriol, halo-nitroso-estriol, halo-nitro-equilenin, halo-nitroso-equilenin, halo-nitro-equilin and halo-nitroso-equilin, halo-nitro-stilbestrol, halo-nitroso-stilbestrol find use as effective anti-tumor agents for treating breast cancer. The estrogen molecules specifically target the active nitro or nitroso moieties to the cancerous breast cells. As indicated above, these cancerous cells are specifically inactivated.
It is therefore a primary object of this invention to provide stable, soluble, non-toxic, highly effective antineoplastic and antiviral drugs. Halo nitro compounds including halo-nitro-estradiol, halo-nitroso-estradiol, halo-nitro-estrone, halo-nitroso-estrone, halo-nitro-estriol, halo-nitroso-estriol, halo-nitro-equilenin, halo-nitroso-equilenin, halo-nitro-equilin, halo-nitroso-equilin, halo-nitro-stilbestrol, halo-nitroso-stilbestrol, 5-iodo-6-nitro-1,2-benzopyrones, 6-nitro-1,2-benzopyrone, 3-nitrobenzamide, 5-nitro-1(2H)-isoquinoline, 7-nitro-1(2H)-isoquinoline, 8-nitro-1(2H)-isoquinoline, 2-nitrobenzamide, 4-nitrobenzamide and their homologues demonstrate these desired properties.